Grantee Research Project Results
2003 Progress Report: Regulation of Embryonic Neuronal Development by Chemical Mixtures from Brick, NJ
EPA Grant Number: R829359Title: Regulation of Embryonic Neuronal Development by Chemical Mixtures from Brick, NJ
Investigators: Reinisch, Carol L. , Kreiling, Jill , Stephens, Raymond
Current Investigators: Reinisch, Carol L. , Cox, Rachel L. , Kreiling, Jill
Institution: Marine Biological Laboratory
EPA Project Officer: Hahn, Intaek
Project Period: July 1, 2001 through June 30, 2004 (Extended to September 23, 2006)
Project Period Covered by this Report: July 1, 2002 through June 30, 2003
Project Amount: $749,333
RFA: Complex Chemical Mixtures (2000) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management , Safer Chemicals
Objective:
The objective of this research project is to use the Spisula solidissima (surf clam) embryo model to examine the effect of environmental toxicants on gene expression and neuronal development.
Progress Summary:
Defining the p73 Gene in Spisula
During Year 1 of the project, we completed sequencing of the p63/p73 gene in Spisula (Ssp63/73) and found that there are two splice variants of the Ssp63/73 gene in Spisula embryos that code for a protein with a molecular weight of approximately 68 kD. These sequences are now published accession numbers AY289767 and AY289768. The nucleotide sequences of the two variants are nearly identical up to their stop codons but diverge in their 3’untranslated regions (UTRs). Further analyses led us to conclude that the two unique transcripts, containing 3’ UTRs of variable lengths, represent tandem alternate polyadenylation sites for the Ssp63/73 gene.
Although alternative splicing has been well documented in the p63/73 gene family, this is the first report of alternate polyadenylation site choice as a control point for p63/73 gene expression in any species. To identify specific post-transcriptional, as well as post-translational signals potentially involved in regulation of p63/73-like expression, we compared Ssp63/p73 nucleotide and Ssp63/73 deduced amino acid sequences to corresponding regions of other mammalian and non-mammalian p63 and p73 homologues. Within the Spisula 3’ UTRs, we identified multiple AU-rich elements that may control translation activation. Within the deduced amino acid sequence, we identified potential sites for sumoylation, a post-translational process that has been identified in mammalian p63 and p73 proteins. In addition, our most recent finding is that the p63/73 gene is expressed in Spisula adult neurons. This confirms our Western blot results showing neuronal expression of a 75 kD p63/73 immuno-reactive protein.
In terms of chemical exposures, we have focused on bromoform, chloroform, and tetrachloroethyelene (BCE), substances of high relevance in the study of autism in Brick, New Jersey. This work is funded by the U.S. Environmental Protection Agency’s Science To Achieve
Results (STAR) program. We reported that exposing embryos to the ternary mixture of chemicals (BCE) increases a developmentally expressed isoform of the regulatory subunit of protein kinase A (RII). We further defined this enhancement of RII production by confocal microscopy and Western blot analyses. An antibody specific for an adult isoform of RII in the related mollusc, Mytilus galloprovinciales , was used to investigate the appearance and localization of the RII antigen during development, both normal and with chemical treatment. Beginning around 72 hours of development, embryos treated with the ternary mixture had cell clusters with extraordinarily high concentrations of RII in the region of the developing gill and in the cells innervating the ciliated velar epithelium.
We confirmed that exposure to the ternary chemical mixture leads to an increase in RII expression by Western blotting. Chemical treatment resulted in protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles virtually identical to their control time point counterparts. In Western blotting, the characteristic 54 and 42 kDa RII subunits were detectable by 24 hours and increased in parallel as the embryos developed. When compared to 24 hour control levels, the combined 54/42 kDa RII signal at 96 and 120 hours increased 4.4 (± 0.3 SD)- and 5.8 (± 1.2 SD)-fold, respectively, in the control, and 6.3 (± 1.5 SD)- and 7.6 (± 1.4 SD)-fold, respectively, in the 1000x treated samples (p < 0.05, n=5). Thus, both subunits of this RII isoform are expressed temporally and are enhanced by chemical exposure after 72 hours of development.
p53 Family Member Analyses
Using molecular approaches, we proposed examining the impact of Brick, NJ chemicals on the p53 family of genes. Members of this family include p53, p73, p97, and p120. We specifically examined p73 because it plays an integral role in neurogenic processes during embryo development.
To analyze the expression p73 during embryonic development, we produced a Spisula-specific p73 polyclonal antibody. A peptide corresponding to a highly conserved region ( YEVTRYTFKHTISL), referred to as the “HOMO-domain,” of the Spisula p73 sequence was synthesized and used to immunize two rabbits. The antibody was affinity-purified from the resulting combined antisera. Spisula tissue samples (~ 50 μg of 24-hour embryos, as well as adult neuron and foot) were subjected to protein gel electrophoresis and immunoblotting using the HOMO-domain primary antibody followed by an alkaline-phosphatase labeled secondary antibody. The HOMO domain antibody recognized multiple bands on Western blots of Spisula neuron, embryo, and foot. T he most prominent band in Spisula neurons migrates at approximately 75 kD. In embryos, an immunoactive band at approximately 75 kD barely is discernible, whereas three others at 97, 120, and 140 kD are prominent. From our Spisula embryo p63/73 sequence data (see section 2), we expected to see a more prominent band in the 65-75 kD range on the Spisula embryo Western blot. The lack of a prominent band in that molecular weight range suggests that mRNA processing may control expression of the gene product on a temporal basis. In Spisula foot, the antibody detected a 63 kD protein in addition to the 75 and 97 kD bands, with the 120 and 140 kD bands being barely discernible.
We tested Spisula embryos at various time points for neuronal changes by confocal microscopy using two Spisula p53 family member antibodies. The p53 family member antibodies were designed to the Spisula p73 protein sequence described above and to the Spisula p120 protein sequence (Jessen-Eller, et al., 2002). Embryos were incubated in a high concentration of the antibody overnight and then probed with a rhodamine- labeled secondary antibody. Neither antibody gave specific, localized signals in the embryos. Neither antibody, therefore, was tested further.
Future Activities:
No future activities were reported by the investigators.
References:
Jessen-Eller K, Kreiling JA, Begley GS, Steele ME, Walker CW, Stephens RE, Reinisch CL. A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls. Environmental Health Perspectives 2002;110(4):377-385.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 2 publications | 1 publications in selected types | All 1 journal articles |
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Type | Citation | ||
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Cox RL, Stephens RE, Reinisch CL. p63/73 homologues in surf clam: novel signaling motifs and implications for control of expression. Gene 2003;320:49-58. |
R829359 (2003) R829359 (2004) |
not available |
Supplemental Keywords:
Spisula solidissima , surf clam, environmental toxicants, p63/73 gene, chemical exposures, protein kinase A, RII, ecology and ecosystems, chemical kinetics, genotoxicity, embryonic neuronal development,, RFA, Scientific Discipline, Waste, Geographic Area, Environmental Chemistry, State, chemical mixtures, Fate & Transport, Hazardous Waste, Ecology and Ecosystems, Hazardous, complex mixtures, contaminated sediments, fate and transport, embryonic neuronal development, fate and transport , effects assessment, biodegradation, bioavailability, hazardous organic substances, New Jersey (NJ), environmental transport and fate, chemical kinetics, genotoxicity, hazardous chemicals, PCE, contaminated soils, analytical modelsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.